Fig. 3

The RBD-specific cellular immune responses elicited by the CW-rBCG::RBD compared with BCG and RBD^AS01. Single-cell suspensions from spleens, inguinal lymph nodes, and lungs were pooled from six mice per group and divided into four replicates for assays at week 12. Cells were restimulated ex vivo with 2.5 µg/ml SARS-CoV-2 RBD peptide pool for 44 h, followed by flow cytometry to assess Tfh and Tcm responses. (a) Immunization schedule and experimental grouping. (b) Detection of Tfh cells (CD4⁺CD44⁺CD62L^-GL7⁺ CXCR5⁺PD-1⁺) in boosted mice. Gating strategy and representative plots shown in Supplementary Fig. S5a–b. (c) CD4⁺Tcm (CD4⁺CD44⁺CD62L^-) detection in the boosted group and (d) primed group; gating shown in Supplementary Fig. S4c. (e) IFN-γ–secreting cells per 10⁵ lung cells measured by ELISPOT. Two wells represent data from two independent experiments, statistical analysis was performed using a t- test. In (b)–(d), data are from two independent experiments, each with four replicates. Data are presented as mean ± SEM. Statistical analysis was performed using an one-way ANOVA, with significance indicated as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.